WO2024157891A1 - 細胞含有容器 - Google Patents

細胞含有容器 Download PDF

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Publication number
WO2024157891A1
WO2024157891A1 PCT/JP2024/001432 JP2024001432W WO2024157891A1 WO 2024157891 A1 WO2024157891 A1 WO 2024157891A1 JP 2024001432 W JP2024001432 W JP 2024001432W WO 2024157891 A1 WO2024157891 A1 WO 2024157891A1
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Prior art keywords
cell
cells
culture
cancer
cell layer
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PCT/JP2024/001432
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English (en)
French (fr)
Japanese (ja)
Inventor
圭 塚本
瑞穂 鈴木
史朗 北野
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Toppan Holdings Inc
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Toppan Holdings Inc
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Priority to EP24747219.4A priority Critical patent/EP4656712A1/en
Priority to JP2024573019A priority patent/JPWO2024157891A1/ja
Publication of WO2024157891A1 publication Critical patent/WO2024157891A1/ja
Priority to US19/279,768 priority patent/US20250346845A1/en
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    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M21/00—Bioreactors or fermenters specially adapted for specific uses
    • C12M21/08—Bioreactors or fermenters specially adapted for specific uses for producing artificial tissue or for ex-vivo cultivation of tissue
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/06—Tubular
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M23/00—Constructional details, e.g. recesses, hinges
    • C12M23/02—Form or structure of the vessel
    • C12M23/10—Petri dish
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12M—APPARATUS FOR ENZYMOLOGY OR MICROBIOLOGY; APPARATUS FOR CULTURING MICROORGANISMS FOR PRODUCING BIOMASS, FOR GROWING CELLS OR FOR OBTAINING FERMENTATION OR METABOLIC PRODUCTS, i.e. BIOREACTORS OR FERMENTERS
    • C12M35/00—Means for application of stress for stimulating the growth of microorganisms or the generation of fermentation or metabolic products; Means for electroporation or cell fusion
    • C12M35/08—Chemical, biochemical or biological means, e.g. plasma jet, co-culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0062—General methods for three-dimensional culture
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/0068—General culture methods using substrates
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N5/00—Undifferentiated human, animal or plant cells, e.g. cell lines; Tissues; Cultivation or maintenance thereof; Culture media therefor
    • C12N5/06—Animal cells or tissues; Human cells or tissues
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G—PHYSICS
    • G01—MEASURING; TESTING
    • G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
    • G01N33/00—Investigating or analysing materials by specific methods not covered by groups G01N1/00 - G01N31/00
    • G01N33/48—Biological material, e.g. blood, urine; Haemocytometers
    • G01N33/50—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing
    • G01N33/5005—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells
    • G01N33/5008—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics
    • G01N33/5011—Chemical analysis of biological material, e.g. blood, urine; Testing involving biospecific ligand binding methods; Immunological testing involving human or animal cells for testing or evaluating the effect of chemical or biological compounds, e.g. drugs, cosmetics for testing antineoplastic activity
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2503/00—Use of cells in diagnostics
    • C12N2503/02—Drug screening
    • C—CHEMISTRY; METALLURGY
    • C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
    • C12N2513/00—3D culture

Definitions

  • the present invention relates to a cell-containing vessel. More specifically, the present invention relates to a cell-containing vessel, a method for culturing cells, and a method for evaluating the effect of a drug on a cell.
  • This application claims priority to Japanese Patent Application No. 2023-008762, filed in Japan on January 24, 2023, the contents of which are incorporated herein by reference.
  • cancer research has focused on experiments using established cell lines that have been subcultured under optimized conditions.
  • cancer cell lines that have been maintained and cultured outside of the body for many years have changed in properties compared to the original patient tumor tissue, and it may not be possible to say that they fully reflect in vivo behavior. Therefore, primary culture of cancer cells is seen as promising for developing more precise anticancer drugs and selecting the most appropriate treatment for each patient.
  • Non-Patent Document 1 describes a CD-DST (Collagen gel droplet embedded drug sensitivity test) method using primary cultured cells.
  • This test method is a drug sensitivity test in which tissue or cells isolated from a patient are embedded and cultured in collagen gel for verification.
  • tissue or cells isolated from a patient are embedded and cultured in collagen gel for verification.
  • Methods proposed for primary culture of cancer cells from patient tumor tissue include adding the ROCK inhibitor Y-27632 to the culture medium to inhibit cell death (also called apoptosis) associated with cell dispersion (Non-Patent Document 2), and obtaining cell aggregates of a certain size while maintaining intercellular adhesion and culturing them in suspension (Patent Document 1).
  • a serum-free medium for stem cells is used to which serum substitutes and various growth factors have been added.
  • serum-free medium for stem cells is generally expensive.
  • signal pathways different from those in the actual living body are enhanced or suppressed. In such an environment, there is a possibility that results different from those in the actual living body may be obtained, particularly in sensitivity tests using molecular targeted drugs.
  • Patent Document 2 describes a method for primary culture of cells in tissues (also called biological tissues) collected from a living organism using a culture medium generally used for cell culture, without adding growth factors or any inhibitors.
  • Patent No. 5652809 International Publication No. 2019/039457
  • the present invention aims to provide a technique for long-term culture of primary cells using a general cell culture medium without adding growth factors or any inhibitors.
  • the present invention can also be applied to cells other than primary cells.
  • a cell-containing container comprising: a cell culture container; a cell culture medium contained in the cell culture container; a first cell layer arranged on a cell culture surface of the cell culture container; and a second cell layer arranged on the first cell layer and containing cells constituting the interstitium, wherein the second cell layer is detachable from the first cell layer.
  • the cell-containing container described in [1] wherein the first cell layer contains primary cells.
  • [4] The cell-containing container described in any one of [1] to [3], wherein the second cell layer is gelled.
  • [5] A method for culturing cells, comprising the steps of incubating a cell-containing container described in any one of [1] to [4], and subculturing the cells contained in the first cell layer by replacing the second cell layer.
  • [6] A method for evaluating the effect of a drug on cells, comprising the steps of incubating a cell-containing container described in any one of [1] to [4] in the presence of a drug, replacing the second cell layer and passaging the cells contained in the first cell layer, and evaluating the effect of the drug on the cells contained in the first cell layer.
  • the present invention provides a technique for long-term culture of primary cells using a general cell culture medium without adding growth factors or any inhibitors.
  • the present invention can also be applied to cells other than primary cells.
  • FIG. 1 is a schematic cross-sectional view illustrating the structure of an example of a cell-containing container.
  • FIG. 2 is a schematic cross-sectional view illustrating an example of a method for producing a second cell layer (cell structure) containing cells that constitute the interstitium.
  • the present invention provides a cell-containing container comprising a cell culture container, a cell culture medium contained in the cell culture container, a first cell layer arranged on a cell culture surface of the cell culture container, and a second cell layer including cells constituting the interstitium arranged on the first cell layer, wherein the second cell layer is detachable from the first cell layer.
  • FIG. 1 is a schematic cross-sectional view illustrating the structure of an example of a cell-containing container of this embodiment.
  • the cell-containing container 100 includes a cell culture container 110, a cell culture medium 120 contained in the cell culture container 110, a first cell layer 130 including cells 131 arranged on a cell culture surface 111 of the cell culture container 110, and a second cell layer 140 including cells constituting the interstitium arranged on the first cell layer 130, and the second cell layer 140 is detachable from the first cell layer 130.
  • the cell-containing vessel 100 further includes a tubular member 150 having openings 151, 152 at both ends, and the tubular member 150 is housed in the cell culture vessel 110 such that one opening 151 is in contact with the first cell layer 130, and the second cell layer 140 may be disposed inside the tubular member 150. Details of the tubular member 150 will be described later.
  • the cell-containing container of this embodiment allows primary cells to be cultured for a long period of time using a general cell culture medium without adding growth factors or any inhibitors. That is, the cells 131 contained in the first cell layer 130 may be primary cells or cells other than primary cells. Examples of cells other than primary cells include established cell lines.
  • being able to culture primary cells for a long period of time means that the primary cells can be grown for, for example, 5 days or more, preferably 10 days or more, more preferably 20 days or more, and even more preferably 30 days or more.
  • the cell culture vessel 110 is not particularly limited, and a vessel that is usually used for cell culture can be used.
  • the cell culture vessel 110 may be a dish, a well plate, or the like.
  • a well plate having 6 wells, 12 wells, 24 wells, 48 wells, or 96 wells can be used.
  • the cell culture surface 111 of the cell culture vessel 110 may be a flat bottom, or may have a lattice-shaped or honeycomb-shaped uneven structure formed thereon.
  • the tubular member 150 is not particularly limited as long as it is capable of constructing a second cell layer 140 (hereinafter sometimes referred to as a "cell structure") containing cells that constitute the interstitium and capable of culturing the constructed cell structure, and for example, a cell culture insert (e.g., a Transwell (registered trademark) insert, a Netwell (registered trademark) insert, a Falcon (registered trademark) cell culture insert, or a Millicell (registered trademark) cell culture insert, etc.), a tube, a pipe, etc. can be used.
  • a cell culture insert e.g., a Transwell (registered trademark) insert, a Netwell (registered trademark) insert, a Falcon (registered trademark) cell culture insert, or a Millicell (registered trademark) cell culture insert, etc.
  • a tube, a pipe, etc. can be used.
  • the tubular member 150 has openings at both ends. At least a portion of the contents of the tubular member 150 (e.g., components secreted from cells) can pass through the openings to the outside of the tubular member 150.
  • a semipermeable membrane also called a membrane
  • a cell culture insert may be used as the tubular member 150 after removing the membrane that is usually placed on the bottom surface of the insert.
  • the upper part of the cylindrical member 150 may have a handle for attaching and detaching the cylindrical member 150 from a dish or well plate.
  • a magnet may also be provided on the upper part of the cylindrical member 150 so that the cylindrical member 150 can be retrieved by magnetic force.
  • the area of the cross section perpendicular to the axial direction of the tubular member 150 must be smaller than the bottom area of the cell culture vessel 110 (also referred to as the area of the cell culture surface 111) and must be able to be accommodated inside the cell culture vessel 110.
  • the tubular member 150 is accommodated in the cell culture vessel 110 so that one opening 151 contacts the cell culture surface 111 of the cell culture vessel 110.
  • the tubular member 150 is accommodated in the cell culture vessel 110 so that one opening 151 contacts the first cell layer 130 arranged on the cell culture surface 111.
  • Cell culture medium In the cell culture vessel of this embodiment, a general cell culture medium that does not contain growth factors or inhibitors of any signal transduction pathways can be used as the cell culture medium 120.
  • the medium is not particularly limited, and examples thereof include media in which serum is added at about 1 to 20% by volume to basic media such as DMEM, EMEM, MEM ⁇ , RPMI-1640, McCoy's 5A, and Ham's F-12.
  • serum include calf serum (CS), fetal bovine serum (FBS), and fetal horse serum (HBS).
  • Primary cells refer to cells directly collected from biological tissue. Primary cells are thought to closely reflect the properties of the biological tissue from which they are derived. Culturing primary cells is called primary culture. In primary culture, multiple types of cells contained in biological tissue may be cultured simultaneously, or only a specific type of cell may be isolated and cultured from the cells contained in biological tissue.
  • the biological tissue from which the primary cells are derived may be tissue collected from any animal species.
  • biological tissue collected from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, or rats can be used.
  • the biological tissue may be solid or liquid.
  • solid tissue include epithelial tissue, connective tissue, muscle tissue, nerve tissue, interstitial tissue, and mucosal tissue that have been surgically excised.
  • liquid tissue include body fluids such as blood, lymph, pleural effusion, peritoneal fluid, cerebrospinal fluid, tears, saliva, and urine.
  • These tissues can be extracted with a scalpel or laser, or collected with a syringe or swab, during surgery or endoscopic examination, for example.
  • Human biological tissues that have been collected for clinical examinations, for example, can be used.
  • Primary cells may be cells derived from normal tissue, or may be cells that have become dysfunctional, such as diseased tissue.
  • cancer cells contained in tumor tissue collected from a cancer patient can be efficiently primary cultured.
  • cancer cells are cells that are derived from somatic cells and have acquired the ability to proliferate indefinitely.
  • primary culture cancer cells may be primary cultured together with cells other than cancer cells contained in tumor tissue collected from a cancer patient, or only cancer cells may be isolated and primary cultured.
  • Cancer cell isolation methods may be any commonly used method, including separation using a cell sorter, magnetic separation, dielectrophoresis, size fractionation, and density gradient fractionation. One of these methods may be used, or two or more methods may be used in combination.
  • the cell isolation method may be appropriately determined based on the organ from which the original patient's tumor originated, the clinical background, or the results of various previous tests.
  • cancer cells When using a cell sorter, cancer cells can be selected by staining them with fluorescently labeled antibodies or fluorescent probes, and then separating out the cells that are positive for the staining process. Cell sorters can also distinguish between live and dead cells from the values of forward and side scattered light, making it possible to more efficiently select and recover live cancer cells.
  • cells When using magnetic separation, cells are magnetically labeled using antibodies, and any method can be selected, such as the positive selection method, in which labeled cancer cells are recovered by magnetism, or the negative selection method, in which cells other than labeled cancer cells are removed by magnetism.
  • disease-related cells collected from patients suffering from various diseases can be primary cultured with a high success rate.
  • the obtained primary culture of disease-related cells is particularly suitable for cell-based assays.
  • the cell-containing container of this embodiment is also useful for establishing culture lines of disease-related cells collected from patients. For example, by primary culture of cells contained in a patient's tumor tissue using the cell-containing container of this embodiment, it is possible to efficiently establish a patient-derived cancer cell line that reflects the characteristics of the patient's tumor, such as proliferation ability, better than a normal cell line.
  • Cancers from which the cancer cells for primary culture are derived include, for example, breast cancer (e.g., invasive ductal carcinoma, ductal carcinoma in situ, and inflammatory breast cancer, etc.), prostate cancer (e.g., hormone-dependent prostate cancer and hormone-independent prostate cancer, etc.), pancreatic cancer (e.g., and pancreatic ductal carcinoma, etc.), gastric cancer (e.g., papillary adenocarcinoma, mucinous adenocarcinoma, and adenosquamous carcinoma, etc.), lung cancer (e.g., non-small cell lung cancer, small cell lung cancer, and malignant mesothelioma, etc.), colon cancer (e.g., gastrointestinal stromal tumor, etc.), rectal cancer (e.g., gastrointestinal stromal tumor, etc.) tumors, etc.), colon cancer (e.g., familial colorectal cancer, hereditary nonpolyposis colorectal cancer, and gastrointestinal
  • the biological tissue is a solid tissue
  • Mechanical methods using scissors, knives, scalpels, tweezers, etc. are preferably used to fragment the biological tissue, but are not limited to these. It is preferable to fragment the biological tissue to about 5 mm or less, for example, so that the primary cells inside can be extracted more efficiently.
  • Sliced biological tissue can be used as is for primary culture, but it is also preferable to subject it to enzyme treatment. Enzyme treatment makes it easier for the primary cells present inside the slicing material to be exposed to the surface, making them more likely to come into contact with the cell structure. Enzyme treatment can also be performed when the biological tissue is liquid tissue.
  • the enzyme used in the enzymatic treatment of the biological tissue or its fragments is not particularly limited, but an enzyme that decomposes proteins, sugars, lipids, nucleic acids, etc. is preferably used.
  • the enzyme used in the enzymatic treatment of the fragments of biological tissue may be one type or two or more types.
  • it is preferable to use one or more enzymes selected from the group consisting of trypsin, collagenase, dispase, elastase, papain, and hyaluronidase it is more preferable to use collagenase or two or more enzymes including collagenase, and it is even more preferable to use collagenase and dispase together with other enzymes as necessary.
  • the enzyme used is not particularly limited as long as it has the desired enzymatic activity, and may be an enzyme derived from any biological species, or may be an artificial enzyme obtained by modifying a naturally occurring enzyme. It may also be extracted and purified from various cells, or may be chemically synthesized.
  • DNase I may be used in combination to prevent cells from clumping together due to the effect of DNA released from cells lysed during the fragmentation or enzymatic treatment.
  • the DNase I used is not particularly limited as long as it is an enzyme with DNase I activity. Examples of commercially available enzyme mixes that contain DNase I and enzymes that degrade biological components such as proteins include Liberase Blendzyme 1 (registered trademark) (manufactured by Roche Diagnostics) and Tumor Dissociation Kit (manufactured by Miltenyi Biotec).
  • the temperature of the enzyme treatment may be any temperature at which the enzyme used can exert its enzymatic activity. In order to minimize the effect on the cells in the fragments of biological tissue, the temperature of the enzyme treatment is preferably 30 to 40°C, and more preferably 37°C.
  • the treatment time of the enzyme treatment is not particularly limited, and can be, for example, 10 to 90 minutes, and preferably 30 to 60 minutes.
  • the cell count and live cell count can be performed by standard methods.
  • the live cell count can be measured by a staining method using trypan blue.
  • the fragmented biological tissue may be washed with a buffer solution or culture medium prior to the enzyme treatment.
  • the buffer solution used for washing may be a phosphate buffer solution, an acetate buffer solution, a citrate buffer solution, a borate buffer solution, a tartrate buffer solution, a Tris buffer solution, or PBS.
  • Antibiotics may also be added to the buffer solution used for washing or the culture medium.
  • the tissue may be washed with phosphate buffered saline (PBS) containing penicillin G (200 U/mL), streptomycin sulfate (200 ⁇ g/mL), and amphotericin B (0.5 ⁇ g/mL).
  • PBS phosphate buffered saline
  • the number of washes may be determined appropriately depending on the origin of the collected biological tissue, but 3 to 8 times is preferable. Washing with a buffer solution or culture medium may be performed only after the enzyme treatment, or before or after the enzyme treatment.
  • only primary cells of the desired cell type are selected from the biological tissue or its enzyme-treated product, and then these selected primary cells are seeded on the cell culture surface 111 of the cell culture vessel 110.
  • only the cancer cells are seeded on the cell culture surface 111 of the cell culture vessel 110.
  • cancer cells can be selected using the expression of cancer cell-specific proteins or increased enzyme activity as cancer markers.
  • proteins specifically expressed in cancer cells such as EpCAM, CEA, Cytokeratin, or HER2
  • cancer cells can be visualized by immunohistochemistry (IHC) staining or immunofluorescence (IF) staining using antibodies against these.
  • enzyme activity of ⁇ -glutamyl transpeptidase or ⁇ -galactosidase which is increased in cancer cells
  • these enzyme activities can be measured using fluorescent probes such as ProteoGREEN (registered trademark, manufactured by Goryo Chemical Co., Ltd.) or GlycoGREEN (registered trademark, manufactured by Goryo Chemical Co., Ltd.).
  • cells 131 are seeded on the cell culture surface 111 of the cell culture vessel 110 and incubated to obtain a first cell layer 130 containing the cells 131.
  • the cells constituting the stroma (also called interstitial cells) constituting the second cell layer 140 are not particularly limited, and may be cells collected from an animal, cells obtained by culturing cells collected from an animal, cells obtained by subjecting cells collected from an animal to various treatments, or cultured cell lines. In addition, commercially available cells or cells derived from a patient may be used. In the case of cells collected from an animal, the site of collection is not particularly limited, and the cells may be somatic cells derived from bone, muscle, internal organs, nerves, brain, bone, skin, blood, or the like, germ cells, or embryonic stem cells (ES cells).
  • ES cells embryonic stem cells
  • the species from which the cells constituting the cell structure according to the present invention are derived is not particularly limited, and cells derived from animals such as humans, monkeys, dogs, cats, rabbits, pigs, cows, mice, or rats can be used.
  • the cells obtained by culturing cells collected from an animal may be primary culture cells or subculture cells.
  • examples of cells subjected to various treatments include induced pluripotent stem cells (iPS cells) and cells after differentiation induction.
  • the cell structure may be composed only of cells derived from the same biological species, or may be composed of cells derived from multiple types of biological species.
  • interstitial cells examples include endothelial cells, fibroblasts, pericytes, immune cells, nerve cells, mast cells, epithelial cells, cardiac muscle cells, hepatic cells, pancreatic islet cells, tissue stem cells, and smooth muscle cells.
  • Immune cells are cells involved in immunity. Specific examples include lymphocytes, macrophages, and dendritic cells. Lymphocytes include T cells, B cells, NK cells, and plasma cells.
  • the interstitial cells contained in the cell structure according to the present invention may be of one type or of two or more types.
  • the interstitial cells contained in the cell structure according to the present invention preferably include one or more types selected from the group consisting of fibroblasts, pericytes, endothelial cells, and immune cells.
  • the number of interstitial cells in the cell structure is not particularly limited, but in order to produce a cell structure that more closely resembles interstitial tissue, the ratio of interstitial cells to all cells constituting the cell structure (i.e., the ratio of the number of endothelial cells to the total number of cells constituting the cell structure) is preferably 30% or more, more preferably 50% or more, even more preferably 70% or more, and particularly preferably 80% or more.
  • the upper limit of the ratio of interstitial cells to all cells constituting the cell structure may be, for example, 100%.
  • vascular network structure and a lymphatic network structure are important for a cell structure to exhibit functions similar to those of interstitial tissue in a living body. For this reason, a cell structure having a vascular network structure is preferable.
  • the vascular network structure may be formed only inside the cell structure, or may be formed so that at least a part of it is exposed on the surface or bottom surface of the cell structure.
  • the vascular network structure may be constructed throughout the cell structure, or may be formed only in a specific cell layer.
  • the term "vascular network structure” refers to a net-like structure such as a vascular network or lymphatic network in a living tissue.
  • the vascular network structure can be formed by including endothelial cells that form blood vessels as interstitial cells.
  • the endothelial cells included in the cell structure may be vascular endothelial cells or lymphatic endothelial cells.
  • the cell structure may also include both vascular endothelial cells and lymphatic endothelial cells.
  • the cells other than endothelial cells in the cell structure are preferably cells that constitute the tissue surrounding the blood vessels in the living body, since endothelial cells tend to form a vascular network that retains its original function and shape. Since this is closer to the interstitial tissue and its surrounding environment in the living body, it is more preferable that the cell structure contains at least fibroblasts as the cells other than endothelial cells, and it is even more preferable that the cell structure contains vascular endothelial cells and fibroblasts, lymphatic endothelial cells and fibroblasts, or vascular endothelial cells, lymphatic endothelial cells and fibroblasts.
  • the cells other than endothelial cells contained in the cell structure may be cells derived from the same biological species as the endothelial cells, or may be cells derived from a different biological species.
  • the number of endothelial cells in the cell structure is not particularly limited as long as it is sufficient to form a vascular network structure, and can be appropriately determined taking into consideration the size of the cell structure, the cell types of endothelial cells and cells other than endothelial cells, etc.
  • a cell structure in which a vascular network structure is formed can be prepared by setting the abundance ratio of endothelial cells to all cells constituting the cell structure (i.e., the ratio of the number of endothelial cells to the total number of cells constituting the cell structure) to 0.1% or more.
  • the number of endothelial cells in the cell structure is preferably 0.1% or more of the number of fibroblasts, and more preferably 0.1 to 5.0%.
  • the total number of vascular endothelial cells and lymphatic endothelial cells is preferably 0.1% or more of the number of fibroblasts, and more preferably 0.1 to 5.0%.
  • the size and shape of the cell structure are not particularly limited. Since a cell structure closer to the interstitial tissue in the living body can be formed and primary culture can be expected in an environment closer to that in the living body, the thickness of the cell structure is preferably 5 ⁇ m or more, more preferably 30 ⁇ m or more, even more preferably 100 ⁇ m or more, and particularly preferably 150 ⁇ m or more. The thickness of the cell structure is preferably 500 ⁇ m or less, more preferably 400 ⁇ m or less, and even more preferably 200 ⁇ m or less. The upper and lower limits of the thickness of the cell structure can be arbitrarily combined.
  • the thickness of the cell structure may be 5 ⁇ m or more and 500 ⁇ m or less, more preferably 30 ⁇ m or more and 400 ⁇ m or less, 100 ⁇ m or more and 200 ⁇ m or less, or 150 ⁇ m or more and 200 ⁇ m or less.
  • the second cell layer 140 (i.e., cell structure) containing cells constituting the interstitium may be a structure consisting of a single or multiple cell layers containing interstitial cells, and the method of manufacturing the second cell layer 140 is not particularly limited.
  • the second cell layer 140 may be manufactured by sequentially stacking cells containing interstitial cells one by one, or two or more cell layers may be manufactured at once, or multiple cell layers may be manufactured by appropriately combining these methods.
  • the cell structure may be a multi-layer structure in which each cell layer is made up of a different cell type, or the cell type constituting each cell layer may be common to all layers of the structure.
  • a cell structure may be manufactured by forming a layer for each cell type and stacking these cell layers in sequence.
  • a cell mixture containing multiple types of cells may be prepared in advance, and a multi-layered cell structure may be manufactured from this cell mixture all at once.
  • a method for producing a cell structure by sequentially stacking layers one by one is, for example, the method described in Patent No. 4919464, in which a step of forming a cell layer and a step of contacting the formed cell layer with a solution containing ECM (also called extracellular matrix) components are alternately repeated to continuously stack cell layers.
  • ECM also called extracellular matrix
  • a cell structure in which a vascular network structure is formed throughout the entire cell structure can be produced by preparing a cell mixture in which all the cells that make up the cell structure are mixed in advance, and forming each cell layer with this cell mixture.
  • a cell structure in which a vascular network structure is formed only in the layer consisting of endothelial cells can be produced by forming each cell layer for each cell type.
  • An example of a method for constructing two or more cell layers at once is the method described in Japanese Patent No. 5850419.
  • the entire surface of a cell is coated in advance with a polymer containing an arginine-glycine-aspartic acid (RGD) sequence to which integrins bind, and a polymer that interacts with the polymer containing the RGD sequence, and the coated cells coated with this adhesive film are placed in a container, and then the coated cells are accumulated by centrifugation or the like, to produce a cell structure consisting of multiple cell layers.
  • a cell mixture is prepared in advance by mixing all the cells that make up the cell structure, and an adhesive component is added to this cell mixture to prepare the coated cells. This makes it possible to produce a cell structure with a homogenous cell composition throughout the entire structure by a single centrifugation process.
  • the second cell layer 140 (i.e., cell structure) containing cells constituting the interstitium can be produced by a method including the steps of: (a) obtaining a mixture containing cells including interstitial cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte; (b) gelling the mixture to obtain a gel composition; and (c) incubating the gel composition to obtain a cell structure. Each step is described below.
  • step (a) a mixture containing the above-mentioned cells including interstitial cells, a cationic substance, an extracellular matrix component, and a polyelectrolyte is obtained.
  • the cells including interstitial cells, the cationic substance, the extracellular matrix component, and the polyelectrolyte may be mixed in an aqueous solvent.
  • the aqueous solvent include water, a buffer solution, and a culture medium.
  • any positively charged substance can be used as long as it does not adversely affect cell growth.
  • the cationic substance include cationic buffers such as Tris-hydrochloric acid, Tris-maleic acid, Bis-Tris, and HEPES, ethanolamine, diethanolamine, triethanolamine, polyvinylamine, polyallylamine, polylysine, polyhistidine, and polyarginine, but are not limited to these.
  • cationic buffers are preferred, and Tris-hydrochloric acid is more preferred.
  • the concentration of the cationic substance in the mixture in step (a) is not particularly limited as long as it does not adversely affect cell growth.
  • the concentration of the cationic substance is preferably 10 to 100 mM, and may be, for example, 20 to 90 mM, for example, 30 to 80 mM, for example, 40 to 70 mM, or for example, 45 to 60 mM.
  • the pH of the cationic buffer is not particularly limited as long as it does not adversely affect cell growth.
  • the pH of the cationic buffer is preferably 6.0 to 8.0.
  • the pH of the cationic buffer may be 7.0, 7.1, 7.2, 7.3, 7.4, 7.5, 7.6, 7.7, 7.8, 7.9, or 8.0.
  • the pH of the cationic buffer is more preferably 7.2 to 7.6, and even more preferably about 7.4.
  • any component constituting the extracellular matrix can be used as long as it does not adversely affect cell growth.
  • the extracellular matrix component include, but are not limited to, collagen, laminin, fibronectin, vitronectin, elastin, tenascin, entactin, fibrillin, proteoglycan, and combinations thereof.
  • the extracellular matrix component may be a modified or variant of the above-mentioned components.
  • the extracellular matrix component may be used alone or in combination of two or more types.
  • Proteoglycans include chondroitin sulfate proteoglycans, heparan sulfate proteoglycans, keratan sulfate proteoglycans, and dermatan sulfate proteoglycans.
  • extracellular matrix components collagen, laminin, and fibronectin are preferred, with collagen being particularly preferred.
  • the total content of the extracellular matrix components in the mixture in step (a) is not particularly limited as long as it does not adversely affect cell growth, and may be 0.005 mg/mL to 1.5 mg/mL, 0.005 mg/mL to 1.0 mg/mL, 0.01 mg/mL to 1.0 mg/mL, 0.025 mg/mL to 1.0 mg/mL, or 0.025 mg/mL to 0.1 mg/mL.
  • the extracellular matrix components can be dissolved in an appropriate solvent before use. Examples of the solvent include, but are not limited to, water, a buffer solution, and acetic acid. Among these, a buffer solution or acetic acid is preferred.
  • polymer electrolyte refers to a polymer having a dissociable functional group in the polymer chain.
  • any polymer electrolyte can be used as long as it does not adversely affect cell growth.
  • polymer electrolyte examples include, but are not limited to, glycosaminoglycans such as heparin, chondroitin sulfate (e.g., chondroitin 4-sulfate and chondroitin 6-sulfate), heparan sulfate, dermatan sulfate, keratan sulfate, and hyaluronic acid; dextran sulfate, rhamnan sulfate, fucoidan, carrageenan, polystyrene sulfonic acid, polyacrylamide-2-methylpropane sulfonic acid, polyacrylic acid, and combinations thereof.
  • the polymer electrolyte may be a derivative of the above. These polymer electrolytes may be used alone or in combination of two or more.
  • the polymer electrolyte is preferably a glycosaminoglycan.
  • heparin, chondroitin sulfate, and dermatan sulfate are preferred, and heparin is particularly preferred.
  • the concentration of the polyelectrolyte in the mixture in step (a) is not particularly limited as long as it does not adversely affect cell growth. Unlike extracellular matrix components, the polyelectrolyte is effective at any concentration up to the solubility limit, and does not inhibit the effects of the extracellular matrix components.
  • the concentration of the polyelectrolyte is preferably 0.005 mg/mL or more, and may be 0.005 mg/mL to 1.0 mg/mL or less, 0.01 mg/mL to 1.0 mg/mL or less, 0.025 mg/mL to 1.0 mg/mL or less, or 0.025 mg/mL to 0.1 mg/mL or less.
  • the polymer electrolyte may be dissolved in a suitable solvent.
  • suitable solvent include, but are not limited to, water and buffer solutions.
  • a cationic buffer solution is used as the cationic substance
  • the polymer electrolyte may be dissolved in the cationic buffer solution.
  • the blending ratio (final concentration ratio) of the polymer electrolyte to the extracellular matrix component in the mixture in step (a) is preferably 1:2 to 2:1, may be 1:1.5 to 1.5:1, or may be 1:1.
  • the cells including the interstitial cells, the cationic substance, the extracellular matrix components, and the polyelectrolyte can be mixed in a suitable container such as a dish, a tube, a flask, a bottle, a well plate, or a cell culture insert. Mixing of these may also be performed in the container used in step (b).
  • the mixture in step (a) may also contain other components in addition to the cells including interstitial cells, the cationic substance, the extracellular matrix components, and the polymer electrolytes.
  • other components include a gelling agent and a cell culture medium necessary for obtaining a gel composition in step (b).
  • Gelling Agent examples include an extracellular matrix component, agarose, pectin, a combination of fibrinogen and thrombin, etc.
  • the gelling agent may be contained in the mixture in step (a) in advance, or may be added to the mixture in step (a) in step (b) described below.
  • step (b) the mixture obtained in step (a) is gelled to obtain a gel composition.
  • the gelling method varies depending on the gelling agent used, and may be, for example, placing the mixture obtained in step (a) under gelling conditions. Alternatively, a gelling agent may be added to the mixture obtained in step (a) and then placed under gelling conditions.
  • the gelling conditions include allowing the mixture obtained in step (a) to stand at about 37°C.
  • the extracellular matrix component contained in the mixture in step (a) gels, and a gel composition is obtained.
  • an extracellular matrix component may be further added to the mixture obtained in step (a), and the mixture may be allowed to stand at about 37°C to gel.
  • agarose when agarose is used as a gelling agent, agarose may be added to the mixture obtained in step (a), the agarose may be dissolved at a temperature equal to or higher than the melting point of the agarose used, and the mixture may be allowed to stand at a temperature equal to or lower than the solidifying point of the agarose used to cause gelation.
  • pectin When pectin is used as a gelling agent, pectin may be added to the mixture obtained in step (a). As a result, the pectin gels due to divalent ions such as calcium ions contained in the mixture, and a gel composition is obtained.
  • Fibrinogen and thrombin may also be used as gelling agents.
  • Fibrin monomers are formed when thrombin, a type of serine protease, cleaves fibrinogen.
  • the fibrin monomers polymerize with each other under the action of calcium ions to form poorly soluble fibrin polymers.
  • fibrin polymers are cross-linked by the action of factor XIII, a fibrin stabilizing factor, to form mesh-like fibers called stabilized fibrin, which cause blood coagulation.
  • factor XIII a fibrin stabilizing factor
  • a gel composition gelled by fibrin polymers may be referred to as fibrin gel.
  • the step (b) of obtaining a gel composition may include a step of mixing thrombin and fibrinogen with the mixture obtained in step (a).
  • the gel composition in step (b) may contain fibrin gel as a gel component.
  • the step (b) of obtaining a gel composition includes a step (b1) of adding thrombin to the mixture obtained in step (a), and a step (b2) of adding fibrinogen to the mixture to which thrombin has been added, thereby forming a fibrin gel and gelling the mixture.
  • the fibrinogen concentration in the mixture in step (b) is preferably 0.5 mg/mL or more and 25 mg/mL or less.
  • the concentration is 0.5 mg/mL or more, it is more likely to gel when mixed with thrombin.
  • the concentration is 25 mg/mL or less, it is more likely to dissolve in the mixture.
  • the thrombin is dissolved or dispersed in the mixture in step (b).
  • step (c) the gel composition obtained in step (b) is incubated to obtain a cell structure.
  • the time for incubating the gel composition to obtain the cell structure may be 5 minutes to 72 hours.
  • Step (c) has the effect of promoting adhesion between the cells contained in the gel composition, resulting in a stable cell structure.
  • step (c) may be the same as the container used in step (b).
  • the container used in step (b) may be used as is, or the contents may be transferred to a different container.
  • the cell culture in step (c) can be carried out under culture conditions suitable for the cells to be cultured.
  • the medium is not particularly limited, but examples include media in which serum is added to basal media such as DMEM, EMEM, MEM ⁇ , RPMI-1640, McCoy's 5A, and Ham's F-12 at about 1 to 20% by volume.
  • serum include calf serum (CS), fetal bovine serum (FBS), and fetal horse serum (HBS).
  • growth factors such as VEGF, EGF, or FGF may be added to the medium.
  • Various conditions such as the temperature and atmospheric composition of the culture environment may also be adjusted to conditions suitable for the cells to be cultured.
  • Step (c) may be performed after steps (a) and (b) are performed two or more times. By repeating steps (a) and (b), a cell structure having multiple layers can be produced. In other words, a cell structure with a large thickness can be produced.
  • steps (a) and (b) can be repeated, using different cell populations each time, to produce a cell structure composed of different types of cells.
  • FIG. 2 is a schematic cross-sectional view illustrating an example of a method for producing a second cell layer (i.e., a cell structure) containing cells that constitute the interstitium.
  • a cylindrical member 150 is placed inside a suitable container 210.
  • One of the openings 151 of the cylindrical member is then brought into close contact with the bottom surface 211 of the container 210.
  • a cell culture dish can be used.
  • step (a) the mixture obtained in step (a) is placed inside the cylindrical member 150 and step (b) is carried out.
  • step (b) is carried out.
  • the mixture gels to obtain a gel composition 140'.
  • one side 151 of the opening of the cylindrical member 150 is in close contact with the bottom surface 211 of the container 210, so the mixture does not leak out from between the cylindrical member 150 and the bottom surface 211.
  • step (c) adhesion between the cells contained in the gel composition 140' is promoted, resulting in a stable cell structure, and a second cell layer 140 (i.e., a cell structure) containing cells that constitute the interstitium is obtained.
  • a second cell layer 140 i.e., a cell structure
  • a membrane may be disposed on one of the openings 151 of the tubular member.
  • the membrane may or may not be removed after the second cell layer 140 is formed.
  • the second cell layer 140 can be easily peeled off from the bottom surface 211 of the container 210 because it has gelled.
  • the second cell layer 140 thus obtained can be placed on the first cell layer 130 containing primary cells 131 that is placed on the cell culture surface 111 of the cell culture container 110.
  • the cell structure containing the primary cells and interstitial cells is in close contact, making it possible to culture the primary cells.
  • a membrane may be placed on one of the openings 151 of the tubular member.
  • the cell structure including the primary cells and the interstitial cells will be in contact with each other via the membrane.
  • the second cell layer 140 may be placed on the first cell layer 130 together with the tubular member 150, or the second cell layer 140 may be removed from the tubular member 150 and placed on the first cell layer 130.
  • the second cell layer 140 can be easily peeled off from the first cell layer 130 during subculture, which will be described later.
  • the second cell layer 140 is detachable from the first cell layer 130.
  • the second cell layer 140 is gelled, it is easy to peel off the second cell layer 140 from the first cell layer 130.
  • the present invention provides a method for culturing cells, comprising the steps of incubating the cell-containing container described above, and replacing the second cell layer with a second cell layer to passage the cells contained in the first cell layer.
  • the culture method of this embodiment includes a step of seeding first cells in a cell culture vessel to obtain a first cell layer arranged on the cell culture surface of the cell culture vessel, a step of arranging a second cell layer containing second cells constituting the interstitium on the first cell layer to obtain a cell-containing vessel containing a cell culture vessel, a cell culture medium, the first cell layer, and the second cell layer, a step of incubating the cell-containing vessel, and a step of passaging the first cells, in which the second cell layer is replaced with a new one in the step of passaging the first cells.
  • the cell culture vessel, cell culture medium, cells, first cell layer, cells constituting the interstitium, and second cell layer, etc. are the same as those described above.
  • the culture method of this embodiment allows primary cells or cells other than primary cells to be cultured using a general cell culture medium without adding growth factors or inhibitors of any signal transduction pathways. Furthermore, as described above, the first cell layer and the second cell layer can be easily peeled off. Therefore, the second cell layer can be easily replaced, and even if the first cells are primary cells, they can be cultured for a long period of time.
  • the frequency of replacement of the second cell layer may be adjusted appropriately depending on the cells being cultured, but if the cells are primary cells, the second cell layer may be replaced, for example, every 2 to 10 days.
  • the present invention provides a method for evaluating the effect of a drug on cells, comprising the steps of incubating the above-mentioned cell-containing container in the presence of a drug, replacing the second cell layer and passaging the cells contained in the first cell layer, and evaluating the effect of the drug on the cells contained in the first cell layer.
  • the culture method of this embodiment allows primary cells or cells other than primary cells to be cultured using a general cell culture medium without adding growth factors or inhibitors of any signal transduction pathways. Therefore, particularly when the cells contained in the first cell layer are primary cells, it is believed that the behavior of the cells can be evaluated in a state closer to that in the living body.
  • the second cell layer can be easily replaced and cells can be cultured for a long period of time, there is less fluctuation in the evaluation due to external environmental factors, and the cells can be cultured and evaluated under more stable conditions.
  • the second cell layer can be easily replaced, the time and effort required for passaging the cells contained in the first cell layer can be significantly reduced. This results in the following effects: less contamination during passaging, a higher cell recovery rate during passaging, and less cell loss.
  • the drug is not particularly limited, and examples thereof include anticancer drugs when the cells contained in the first cell layer are cancer cells.
  • the method of this embodiment can be said to be a drug screening method.
  • examples of drugs that can be used include natural compound libraries, synthetic compound libraries, and existing drug libraries. This screening method makes it possible to screen drugs using cells.
  • the cells contained in the first cell layer may be primary cells, or may be cells other than primary cells.
  • Cells that compose the interstitium were those shown in Table 1 below.
  • Cell culture vessel The cell culture vessels used in the experimental examples described below are shown in Table 3 below.
  • Cell culture medium The cell culture media used in the experimental examples described below are shown in Table 4.
  • the cell layer containing the cells that constitute the stroma i.e., the second cell layer, may be referred to as the "stromal stamp.”
  • stromal stamps A cell layer containing cells constituting the stroma (i.e., a stromal stamp) was prepared by the following procedure: First, a 10 U/mL thrombin-DMEM solution was prepared, and a 10 mg/mL fibrinogen-DMEM solution was prepared.
  • NHDFs and HUVECs having a cell number of 1/10 to 1/5 that of NHDFs were suspended in 50 mM Tris-HCl buffer solution (pH 7.4) containing 0.05 mg/mL heparin and 0.05 mg/mL collagen.
  • the mixture was centrifuged at 1,000 x g for 1 minute at room temperature, and the supernatant was removed.
  • the precipitate was then suspended in a general-purpose medium to obtain a suspension.
  • the fibrinogen solution and the suspension were then mixed at a ratio of 1:1 (v/v).
  • the mixture was then mixed with the thrombin solution at a ratio of 2:1 (v/v).
  • the membrane was removed from the first container (Transwell culture insert) in Table 3 above to obtain a cylindrical member having openings at both ends.
  • the first container was placed on the second container (petri dish) in Table 3 above, and 100 ⁇ L of the above suspension was seeded inside the first container. Next, it was left to stand in a CO2 incubator (37° C., 5% CO2 ) until the suspension gelled.
  • primary cells Preparation of primary cells As primary cells, commercially available fresh frozen breast cancer tissue (Proteogenomics, Inc., 009-01310) and fresh frozen pancreatic cancer tissue (Proteogenomics, Inc., 009-01310) were used. Each tissue was treated with collagenase/dispase, and then passed through a filter with a pore size of 100 ⁇ m, and the passing fraction was used as primary cells.
  • Primary cells commercially available fresh frozen breast cancer tissue (Proteogenomics, Inc., 009-01310) and fresh frozen pancreatic cancer tissue (Proteogenomics, Inc., 009-01310) were used. Each tissue was treated with collagenase/dispase, and then passed through a filter with a pore size of 100 ⁇ m, and the passing fraction was used as primary cells.
  • the cells were fixed and stained for EpCAM by immunofluorescence staining, and the stained cells were counted as primary cells.
  • the proliferation fold of the primary cells was then calculated using the above formula (1).
  • Table 5 below shows the results of culturing primary cells derived from fresh frozen breast cancer tissue.
  • proliferation of primary cells derived from fresh frozen breast cancer tissue was not observed in either the culture using general-purpose medium or the culture using primary culture medium.
  • proliferation of primary cells was observed up to 14 days after the start of culture, but proliferation stopped after 14 days.
  • proliferation of primary cells was observed over time for 21 days after the start of culture, and proliferation efficiency was the highest.
  • Table 6 below shows the results of culturing primary cells derived from fresh frozen pancreatic cancer tissue.
  • general-purpose medium was used in 2D culture, no proliferation of primary cells derived from fresh frozen pancreatic cancer tissue was observed.
  • primary culture medium was used in 2D culture, a slow tendency for primary cells to proliferate was observed up to 21 days after the start of culture, but a decreasing tendency was confirmed thereafter.
  • 3D culture proliferation of primary cells over time was observed up to 21 days after the start of culture, but proliferation stopped thereafter.
  • proliferation of primary cells was observed over time for 28 days after the start of culture, and proliferation efficiency was the highest.
  • the stromal stamp prepared in Experimental Example 1 was then placed on each well. The cells were then cultured in the general-purpose medium shown in Table 4 above.
  • the anticancer drug SN-38 (Selleck, S4908) was added to the medium of each well to a concentration of 0, 0.01, 0.1, 1 or 10 ⁇ M. Then, on the seventh day after the start of the culture, the interstitial stamp was removed and the number of HT29 cells was counted by ATP assay. Then, the cell viability was calculated by the following formula (2) to evaluate the drug efficacy.
  • Cell viability (%) number of cells in the presence of each drug concentration / number of cells in the absence of drug ⁇ 100 (2)
  • ⁇ Culture of cancer cells using 2D culture method and evaluation of anticancer drugs ⁇ HT29 cells were seeded at 1 ⁇ 10 3 cells/well in the third container (24-well plate) in Table 3. Then, the cells were cultured in the general-purpose medium shown in Table 4.
  • the anticancer drug SN-38 (Selleck, S4908) was added to the medium of each well to a concentration of 0, 0.01, 0.1, 1, or 10 ⁇ M. Then, on the seventh day after the start of culture, the number of HT29 cells was counted by ATP assay. Then, the cell viability was calculated using the above formula (2) to evaluate the drug efficacy.
  • ⁇ Culture of cancer cells using 3D culture method and evaluation of anticancer drugs 2.0 ⁇ 10 6 NHDFs and 3.0 ⁇ 10 4 HUVECs were suspended in an equal mixture of 150 ⁇ L of 0.2 mg/mL heparin/50 mM Tris-HCl buffer (pH 7.4) and 150 ⁇ L of 0.2 mg/mL collagen/5 mM acetic acid solution (pH 3.7). The resulting mixture was centrifuged at room temperature for 1 minute at 1,000 ⁇ g to obtain a viscous body. The resulting viscous body was suspended in DMEM containing 10% FBS.
  • the entire amount of the resulting suspension was seeded in a 24-well Transwell culture insert and centrifuged at room temperature for 1 minute at 400 ⁇ g (gravitational acceleration). This resulted in the formation of a cell layer (i.e., a cell layer containing cells constituting the interstitium) on the membrane of the Transwell culture insert.
  • a cell layer i.e., a cell layer containing cells constituting the interstitium
  • HT29 cells were seeded on the cell layer at 1 ⁇ 103 cells/well and cultured in the general-purpose medium shown in Table 4.
  • the anticancer agent SN-38 (Selleck, S4908) was added to the medium in each well to a concentration of 0, 0.01, 0.1, 1, or 10 ⁇ M.
  • Table 8 below shows the results of the culture evaluation of mouse fertilized eggs. Under conditions using a general-purpose medium, no embryos developed to the blastocyst stage in 2D culture, whereas when using the interstitial stamp, the percentage of embryos that reached the blastocyst stage was 60% when the interstitial stamp used in Experimental Example 1 was used, and 80% when the interstitial stamp was made with the cell composition for fertilized egg culture, the same as with the dedicated fertilized egg medium. This shows that by using the interstitial stamp, the blastocyst attainment rate is almost the same as with the dedicated fertilized egg medium, even when using a general-purpose medium.
  • IPS cell differentiation The differentiation of IPS cells was induced using stromal stamps, and was compared with that of IPS cells cultured using a conventional 2D culture method.
  • HCF GFP-introduced human cardiac fibroblasts
  • HCM YFP-introduced human cardiac myocytes
  • hMSC-UC human mesenchymal stem cells from umbilical cord matrix
  • hMSC-UC human mesenchymal stem cells from umbilical cord matrix
  • a primary differentiation stromal stamp was prepared using hMSC-AT tissue (model number D10134, manufactured by PROMOCELL). The primary differentiation stromal stamp was placed on each drop gel.
  • a stromal stamp for maintaining stability was prepared by replacing the cell composition with GFP-introduced Human Cardiac Fibroblasts (HCF, model number: D10057, manufactured by PROMOCELL) and YFP-introduced Human Cardiac Myocytes (HCM, model number: D10115, manufactured by PROMOCELL) in the method described in Experimental Example 1.
  • the initial differentiation stromal stamp was removed from each drop gel, and the stabilization stromal stamp was placed on each drop gel to induce and maintain differentiation, and the number of days until the cTNT positive cells reached 80% or more was compared.
  • the evaluation was outsourced to Fukushima Cell Factory.
  • Table 9 below shows the results of evaluation of induction of differentiation into cardiac muscle.
  • the 2D culture method showed no signs of differentiation and could not be maintained, whereas when the interstitial stamp was used, the cTNT positive cells reached 80% or more in 15 ⁇ 3 days.
  • the number of days until the cTNT positive cells reached 80% or more was slightly shorter than in the 2D culture method using a medium specifically designed for induction of cardiac muscle differentiation. This shows that by using the interstitial stamp, the number of cTNT positive cells can reach 80% or more in the same number of days as with existing methods, even when a general-purpose medium is used.
  • the present invention provides a technique for long-term culture of primary cells using a general cell culture medium without adding growth factors or any inhibitors.
  • the present invention can also be applied to cells other than primary cells.

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